EXCEED THE SPACE PROVIDED. _ _ ^^ _ _ ___ _ i The long-range goal of the proposed research is to define the structural elements that comprise the outward-facing, water accessible i ; surface of the CFTR chloride channel and are components of the anion-conducting pore, the gating machinery or both; and to ! incorporate this information into a physically-based model for CFTR channel function that will provide the scientific basis for j designing strategies to enhance or attenuate channel function. Understanding the channel function of CFTR will provide insights into ' three devastating diseases; cystic fibrosis (CF), the most effect common fatal inherited disease in the Caucasian population, secretory ; diarrhea, the leading cause of infant mortality worldwide and polycystic kidney disease. It is thought that at least some of the i membrane-spanning segments (TM's) of CFTR contribute to the formation of the anion-selective conduction path, but little is known 1 about the physical basis for conduction, i.e. the anion-channel interactions that govern anion permeability and binding. In addition, i j altered function in mutant CFTRs indicates that at least some of the TMs may be integral parts of the gating machinery. The role of specific residues or groups of residues in conduction and gating is unknown, and identification of these sites would provide data j critical to the design of high conductance pores or ligands that interact with the pore or the gating machinery or both. We propose to | identify residues and/or TMs that comprise the pore by means of covalent labeling coupled with detailed functional analysis, i j Extensive preliminary data demonstrates that it is possible to covalently label cysteines engineered into the outward-facing surface of i i CFTR, and we propose criteria for identifying, among these, "pore-lining" residues based on the detailed analysis of the impact of ! charge deposition on anion conduction. The specific aims are: 1. To identify, by covalent labeling of engineered cysteines, residues j ; that lie on the outward-facing, water-accessible surface of CFTR. 2. To investigate the mechanisms by which covalent and non ; i covalent modification of engineered cysteines alters the conduction and gating properties of CFTR and thereby, to identify residues I that are likely to lie within the pore or contribute to the gating machinery, or both. 3. To identify locations in the protein where j | engineered cysteines exhibit conformation-dependent accessibility to polar thiol reagents as a function of either mutation-induced changes in channel structure or changes in the gating state of the channel; that may be points at which a covalent or non covalent modification can "switch" the protein from one state to another. 4.To test a dielectric stabilization model for permeation by altering the electrostatic environment within the pore by means of helix substitutions or mutations at locations identified by covalent labeling to be "pore-lining". We will incorporate the results of these proposed studies into an evolving model for CFTR as a polarizable tunnel with an outer vestibule that functions to concentrate permeant anions in the vicinity of an inner, rate-limiting barrier. j i __ _ _.. . _ . __ . ,